Endotoxaemia in Patients with Crohn’s Disease: A Longitudinal Study of Elastase/α1-Proteinase Inhibitor and Limulus-Amoebocyte-Lysate Reactivity

J. Clin. Chem. Clin. Biochem.

Vol. 26, 1988, pp. 117-122

© 1988 Walter de Gruyter & Co.

Berlin · New York

Endotoxaemia in Patients with Crohn's Disease:

A Longitudinal Study of Elastase/di-Proteinase Inhibitor and L/mw/M^-Amoebocyte-Lysate Reactivity

By P. C. Fink, C. Sum de Boutemard, R. Haeckel Zentralkrankenhaus S L-Jürgen-Straße, Bremen and

W. Wellmann

Medizinische Hochschule Hannover, Hannover

(Received June 24/December 29, 1987)

Summary: During intensive care, the severity of gut-derived systemic endotoxaemia in 16 patients with active Crohn's disease was characterized by the simultaneous determination of elastase/ai-proteinase-inhibitor and L/ww/ws-amoebocyte-lysate-reactivity. Using both assays in 15/16 patients the detectable endotoxic activity during the disease course was reflected by a parallelism between elastase/a

proteinase-inhibitor and Limulus- amoebocyte-lysate-reactivity. In 8 gut-irrigated patients, significantly lower elastase/a

proteinase-inhibitor concentrations and L/wi//i/,s-amoebocyte-lysate-reactivities were measured compared with 8 gut-nonirrigated patients. The Crohn's disease activity index and the van Hees activity index as well as the inpatient time were significantly lower in lavage patients compared with gut-nonirrigated patients. It is concluded with that elastase/arproteinase-inhibitor can be used as an alternative to the bioassayed L/mw/w^-amoebocyte-lysate- reactivity in order to characterize systemic endotoxaemia.

Introduction

In patients with Crohn's disease, the Grä/w-negative enterobacteriaceae (Escherichia coli

Salmonella, Pro- teus, Klebsiella, Enterobacter, Serratia, Yersinid) as well as their cell wall constituents, the lipopolysac*

charides (endotoxin), contribute to the inflammatory lesions in the bowel wall (1). These are mainly caused by abnormal and invasive bacterial colonisation (2 — 6). During the acute phase of Crohn's disease, a systemic endotoxaemia (7-* 10) and elevated antibody concentrations directed against Escherichia coli-O serotypes (11,12), and lipid A (13 —15), the toxophor within the lipopolysaccharide molecule (16), were found.

Endotoxin induces the release of elastase and other inflammation triggering substances (cathepsin, colla- genase, myeloperoxidase) from mononuclear phago-

cytes (17, 18). The proteolytic inflammatory effect of elastase is generated when the amount of ai-protein- ase-inhibitor does not suffice to neutralize the elastase activity (19).

Lipopolysaccharides also induce a leukocytosis in pa- tients with Crohn's disease (20,21) concomitantly with a neutrophilia and a lymphocytopenia (22, 23). In the damaged bowel wall a massive infiltration of granu- locyteSj macrqphages, cytotoxic-T-cells, B- and plasma-cells was shown (24—26).

Based upon these inter-related features it was of in- terest to determine elastase/a

proteinase-inhibitor in plasma of lipopolysaccharide-stimulated whole blood from healthy adults and patients with Crohn's disease.

The extent of endotoxaemia in lavage and gut-non- irrigated patients (27) with active Crohn's disease was characterized by the simultaneous application of the

J. Clin. Chem. Clin. Biochem. / Vol. 26,1988 / No. 3

turbidimetric L/raM/itf-amoebocyte-lysate-reactivity test (28, 29) and the elastase/tti-proteinase-inhibitor enzyme-linked-immunosorbent assay (30, 31) as well as by the Crohn's disease activity index (32) and the van Hees activity index (33).

Materials and Methods Specimens

From 16 patients (male/female ratio: 7/9, age range: 17—48 years), with active Crohrfs disease confirmed by clinical, ra- diological, endoscopic and pathohistological features, the course of endotoxaemia was assayed during 14 days of inpatient time. All patients were administered total parenteral nutrition, 9620 kJ/24 h in a volume of 2500 ml through a central venous catheter at the day of admission for at least one week. As a standard therapeutic regimen prednisone and sulphasalazine were given orally to all patients.

For 8 lavage patients, 2 whole gut-irrigations were performed by placing a radiopaque polyvinyl tube into the jejunum (34).

NaCl solution (0.15 mol/1) containing 5-acetyl-salicylic acid was used as lavage fluid at 37 °C in the first week (27).

Serum sodium, potassium, bicarbonate, haemoglobin, haema- tocrit and body weight were determined before, during and after whole gut-irrigation. For randomisation, cards were pre- pared, which indicated to which treatment group the patients were allocated. 8/16 patients allocated to the lavage group gave informed consent before treatment.

From a 59 year-old intensive care patient samples were drawn in 2 h intervals to study the 24 h-profile between elastase/ar proteinase-inhibitor and L/ww/wi-amoebocyte-lysate-reactivity.

Fifty clinically apparent healthy adults (male/female ratio:

23/27, age range: 18—64 years), who were routinely examined by the personnel medical service, served to set up a reference range for elastase/a^rproteinase-inhibitor. Laboratory data for these individuals based on haemogram (erythrocyte and leu- kocyte count, haemoglobin, haematocrit), aspartate amino- transferase, alanine aminotransferase, serum creatinine, urea, hepatitis B surface antigen, anti-hepatitis B surface antibody showed normal values in all cases.

From all patients and healthy adults heparinized (50 USP, Heparin-Novo, lot. No. B 551*13, Νονό-Industries D-6500 Mainz) venous blood was obtained by sterile venepuncture (29).

After centrifugation of whole blood at ISO g for 10 min at 20 °C the plasma supernatants were pipetted into sterile LAS- R-borosilicate glass cuvettes 10 χ 75 mm) (cat. No. RD Οδο- ί 72, Hyland Travenol, D-8000 Munich) and stored at -30 °C until use.

To test the influence of various anticoagulants on the elastase/

arproteinase-inhibitor assay, whole blood from 10 healthy adults was anticoagulated either with heparin, or K2-EDTA, 0.24 mol/1 (cat. No. 03258) or citrate, 0.1 mol/1 (cat. No. 05.268, Sarstedt, D-5223 N mbrecht). After different storage times (10 min, 30 min, 60 min) and incubation temperatures (4 °C, 20 °C, 37 °C) the elastase/a^proteinase inhibitor content in plasma was assayed.

To test the release of elastase into plasma from unstimulated and lipopolysaccharide-stimulated whole blood, additional samples from 10 healthy adults and 5 patients with untreated active Crohrfs disease were obtained. After incubation with 20 μg of lipopolysaccharide (lipopolysaccharide from Esche- richia coli 055:B5, lot. No. 3120 Difco-Lab., Detroit, USA) for 30 min at 37 °C elastase/a,-proteinase-inhibitor was determined in plasma.

Elastase/dt-proteinase-inhibitor assay

The elastase/ctt-proteinase-inhibitor assay (lot. No. 41292105), standards (1 μg/l, 2 μg/l, 4.5 jig/1,11.9 μ§/1) as well as the human control plasmas (106 μg/l, 153 μg/l) were kindly supplied by Dr. S. Neumann (Biochemische Forschung Merck, D^6100 Darmstadt). The working procedure of the elastase/arprotein- ase-inhibitor assay was performed unmodified as described elsewhere (30, 31). ' '

Z/iTWM/MJ-arnoebocyte-lysate-test

Lyophilized L/wM/w-y-amoebocyte-lysate-reagent (lot. No. 52-76- 245, Associates of Cape Cod, Woods Hole, MA, USA) and lipopolysaccharide-standard (Novo-Pyrexal forte, 1.0 mg/1, Pyr- oquant Diagnostik, D-6082 Walldorf) (35) were used for all samples. The turbidimetric assay procedure was performed as described elsewhere (28, 29). The absorbance values at 365 nm expressing the amount of lipopolysaccharide activity in the sample were plotted against the absorbance values of the li- popolysaccharide calibration curve thus expressing the Limulus- amoebocyte-lysate-reactivity in the sample as lipopolysacchar- ide-equivalents.

Statistical analysis

For evaluation of results imprecision within-nm and between^

days, Student's t-test (p < 0.025), multi-variate variation anal- ysis (p < 0.05), coefficient of correlation (r) and Spearmarfs coefficient of rank correlation (rs) were performed as described elsewhere (36).

Results

Effect of various anticoagulants, storage times and temperatures on elastase/c^-pro- teinase-inhibitor

No significantly different (p > 0.05) elastase/cti-pro^

teinase-infaibitor concentrations were found in plasma from heparinized, K

-EDTA- or citrate-containing whole blood samples, stored for 10, 30 or 60 min at 4°C or 20 °C (tab. 1). In contrast, a significantly (p < 0.01) elevated elastase/ctrproteinase inhibitor concentration was determined in plasma from whole blood stored 60 min compared with 10« min or 30 min, at 37 °C.

Elastase/c^-proteinase-inhibitor in plasma of lipopolysaccharide-stimulated whole blood

Increased plasma elastase/arproteinase-inhibitor (p < 0.0005) was found in lipopolysaccharide-stim- ulated whole blood (patients with CroHn's disease:

1154.6 ± 157.3 μ^ healthy adults: 231.5 ± 74.6 μg/l) compared with unstimulated whole blood (pa^

tients

with Crohn's disease: 478.0 ±162.5 μg/l;

healthy adults: 92.1 ± 14.0 μg/l). .,

Table l. Plasma-elastase/arproteinase-inhibitor in whole blood samples (n = 10) from healthy adults containing various anti- coagulants and treated with different storage times and incubation temperatures (x = mean value, s = 1 standard deviation).

Temper- ature conditions

4°C 20 °C 37 °C

Storage times Anti- coagulants

Xs

Xs χs

10 min Heparin 78.327.5 81.825.1 85.025.4

K2-EDTA 78.427.0 82.226.8 85.125.7

Citrate 78.626.7 82.526.9 26.584.5

30 min Heparin 78.525.9 82.825.8 25.685.7

K2-EDTA 77.926.3 84.127.6 85.524.2

Citrate 78.426.0 83.826.0 84.225.4

60 min Heparin

80.826.1 84.626.2 124.8 30.8

K2-EDTA 80.326.8 25.184.9 128.9 26.0

Citrate 80.127.6 84.127.4 131.0 30.4

Characterization of endotoxaemia by elas- tase/ai-proteinase-inhibitor and Limnlus- amoebocyte-lysate-reactivity

Prior to the longitudinal study in patients with Crohn's disease, a 24 h-profile of elastase/a

protein- ase-inhibitor and L/ww/M$-amoebocyte-lysate-reactiv- ity was provided for one septic intensive care patient, who had a positive (Escherichia coli) blood culture and leukocyte counts in the range of 7.2 — 8.7 χ 10

/1-A parallel course (r = 0.79) between elastase/

cti-proteinase-inhibitor and positive Limulus-amoe- bocyte-lysate reactivities was found.

The same parallelism (0.68 > r < 0.99; 0.60 > rs

< 1.00) between elastase/cti-proteinase-inhibitor and Lf'mw/ws-amoebocyte-lysate-reactivity was found for 15/16 lavage- and gut^nonirrigated patients with Crohn's disease. Two representative courses are de- picted in figure 1 and 2.

Lavage patients revealed significantly lower (p <

0.025) mean values of elastase/a^proteinase-inhibitor and Lz/ww/M^amoebocyte-lysate-reactivity at day 6 and 8 than gut-nonirrigated patients (figs. 3, 4). In accordance with the course of elastase/arproteinase- inhibitor and jL/WM/wj'-amoebocyte-lysate-reactivity, the Crohn's disease activity index and van Hees index decreased more rapidly (p < 0.025) in the lavage group (Crohn's disease activity index: 228 ± 39 index points to 86 ± 42 index points; van Hees: 214 ± 33 index points to 157 ± 33 index points) than in gut- nonirrigated patients (Crohn's disease activity index:

240 + 46 index points to 158 ± 53 index points; van Hees: 233 ± 46 index points to 191 ± 28 index points). Patients from the lavage group (25.2 ± 8.5 days) were discharged earlier from the hospital than gut-nonirrigated patients (39.1 ±11.9 days) (p < 0.01).

Discussion

In patients with active inflammatory bowel disease and intestinal mucosal lesions the enteral resorption of lipopolysaccharides results in portal endotoxaemia (37), due to insufficient clearance of lipopolysacchar- ides by the hepatic mononuclear phagocytic system (38, 39). Endotoxin may also reach the blood stream by transmural escape through the peritoneal cavity or propagation via intestinal lymphatics and the ductus thoracicus (8).

A systemic endotoxaemia based on single determi- nations of positive L/mw/w^-amoebocyte-lysate-reac- tivity was found in all patients with active Crohn's disease (8, 9). In contrast, Kruis et al. (14) detected a positive L/raw/itf-amoebocyte-lysate-reactivity only in a minority of patients. The cause of the above men- tioned discrepancy may be the interference of Limu- Λ/j-amoebocyte-lysate-reactivity, with polynucleo- tides, peptidoglycans derived from cell walls of Gram- positive bacteria, plasma macromolecules (HDL, phospholipides, inhibitors) and leukocytic factor(s) (28, 29) as well as Lfww/w^-amoebocyte-lysate-batch variabilities. Thus, single determinations of Limulus- amoebocyte-lysate-reactivity are of limited clinical va- lidity in characterizing "endotoxaemia" (29). There- fore, replicate determinations of the LzAww/w^-amoe- bocyte-lysate-reactivity were performed in order to monitor the patient-specific course of endotoxaemia.

Lipopolysaccharides also effect the release of elastase from leukocytes (18, 19). Free elastase amplifies the inflammatory potency of lipopolysaccharides (21, 40) by cleavage of clotting-, complement- and fibrinolytic- factors, immunoglobulins, transport proteins and plasma protein inhibitors (41 -47) as well as by de- struction of collagen, elastin and proteoglycans (48).

J. Clin. Chem. Clin. Biochem. / Vol. 26,1988 / No. 3

0 2 4 6 8 Whole gut

irrigation

11 14 . 21 Inpotient time [d]

250-

10

200 I

150 |

Ο-Ι

100*

50 ΐ l

Fig. 1. Elastase/ai-proteinase-inhibitor, L/w«/Mj-amoebocyte- lysate-reactivity and clinical data in a gut-irrigated pa- tient with active Crohn's disease during the inpatient time (28 days).

Patient S. B. (22.5 years, female), 7 — 8 diarrhoeas/day, 4 kg body weight loss, chill and fever before hospital admission.

Pathohistological area of inflammation: terminal ileum, colon.

Body weight (kg) Fever ( °C) Erythrocyte sedi- (mm)

mentation rate

Crohn's disease (points) activity index

van Hees activity index (points) Leukocyte count (109/1) Cholinesterase (U/l) Total protein (g/1) Protein fractions

albumin QI -globulin a2-globulin -globulin γ-globulin

Treatment: conservative therapy gations at days 3 and 5.

Elastase/ai -proteinase-inhibitor At hospi- talad- mission

5239 22/48 231 2823.6 58052

0.490.06 0.130.10 and 2 whole0.22

L/ww/itf-amoebocyte-lysate-reactivity ο —D —

Atdis- charge

6137 25/56 118 1505.6 205065

0.570.05 0.110.15 gut-irri-0.12

n

0

4 6 11 14 21^ΊΓ

Inpqtient time td] 28 35J σ>

250 J5

Ιο f

200 S 2 150 f- 100 ·§

Fig. 2. Elastase/a!-proteinase-inhibitor, L/fww/wj-amoebocyte- lysate-reactivity and clinical data in a gut-nonirrigated patient with active Crohn's disease during the inpatient time (38 days).

Patient S.H. (23.5 years, male), 10—12 diarrhoeas/day, 8 kg body weight loss before hospital admission.

Pathohistological area of inflammation: terminal ileum, colon.

Body weight Fever

Erythrocyte sedi- mentation rate Crohn's disease

activity index van Hees activity index Leukocytes

Cholinesterase Total protein Protein fractions

albumin arglobulin a²-globulin -globulin γ-globulin Treatment: conservative

(kg)(°Q (mm) (points) (points) (10'/1) (U/l) (g/1)

therapy.

At hospi- talad- mission

6237 55/80 302 227 120015.9 51

0.500.07 0.120.11 0.20

At dis-charge

7237 10/20 156 1138.9 215066

0.700.04 0.070.10 0.10

Elastase/cti-proteinase-inhibitor

Lzmw/wi-arnoebocyte-lysate-reactivity 0-6

Based upon this pathochemical interrelationship, L/ww/ws-amoebocyte-lysate-reactivity and elastase/tti- proteinase-inhibitor were determined in parallel to characterize the course of endotoxaemia.

The precision data (n = lOKorelastase/ai-proteinase- inhibitor (coefficients of variation: imprecision within- run 3.6%; imprecision between-days 9.0%) and the

obtained reference range (95% percentile: < 106 were in accordance with other authors (30, 31, 49).

The practicability (e. g. influence of anticoagulants, storage- and temperature conditions) of the elastase/

a

-proteinase-inhibitor assay showed that in heparin-

K

-EDTA- or citrate-containing plasma, similar elas-

tase/ai-proteinase-inhibitor concentrations were de-

tectable. These findings were the prerequisite for using

3240 .§220 1200 g 180 l160

t HO g 100

Fig. 3.

///*/"7~/

2l . __,. __ ._J4 6 8

Time of lavage treatment

/Reference range elastose / / /

11 14 21 Inpotient time Id]

'/T* 28

Elastase/cti-proteinase-inhibitor concentrations in pa- tients with Crohn's disease (mean values).

Lavage patients o— o, gut-nonirrigated patients ο — ο

|^20

II t.l

2-Ξ Ο

2 4 6 ⁽ 8

Time of lavage treatment

14 21

Inpatient time [d]

Crohn's disease (mean values).

Lavage patients O—o, gut-nonirrigated patients ο — α

a single specimen for both the elastase/a^proteinase- inhibitor-assay and the turbidimetric L/ww/i/s-amoe- bocyte-lysate-reactivity assay. In agreement with Dus- vvald (50), plasma elastase/a

proteinase-inhibitor in- creased by less than 10% up to 60 min at 4 °C or 20 °C. A significantly elevated plasma elastase/aj-pro- teinase-inhibitor was observed only after a 60 min interval at 37 °C between blood sampling and cen- trifugation. This assay was performed to differentiate between the 37 °C-mediated elastase release and the lipopolysaccharide-mediated release of elastase. In the in vitro studies with a 30 min incubation time, the found elastase/ai-proteinase-inhibitor-concentrations were primarily caused by lipopolysaccharide-stimu- lated leukocytes. Although there was a difference be- tween elastase values of unstimulated and lipopoly- saccharide-stimulated whole blood from healthy adults and patients with Crohn's disease the relative elastase increase was similar for both groups.

Similar results were reported by Duswald (50) and Aasen et al. (51) for healthy adults and dogs respec- tively.

To confirm the in vitro findings, elastase/a

protein- ase-inhibitor and Limw/M,s-amoebocyte-lysate-reactiv- ity were determined in a septic intensive care patient with an Escherichia colt bacteraemia. The observed parallel profile between elastase/a^proteinase-inhibi- tor and Lzmw/wj-amoebocyte-lysate-reactivity for this patient and the patient with active Crohn's disease suggest that although different analytes were deter- mined, both tests measured the underlying endotoxic activity simultaneously in a similar manner. Elastase/

ai-proteinase-inhibitor seemed to be a more adequate marker for the inflammatory disease activity than the leukogram, since in the bacteraemic septic patient

with leukocyte values within the reference range, elas- tase/arproteinase-inhibitor levels were found to be constantly elevated.

The constantly lower elastase/aj-proteinase-inhibitor concentrations and L/ww/w^-amoebocyte-lysate-reac- tivities as well as the more rapid reduction of endo- toxic activity in lavage patients, compared with gut- nonirrigated patients, support the hypothesis that the reduced endotoxaemia in lavage patients was due to a reduction of intestinal lipopolysaccharide biomass.

In agreement with our findings a reduction of intes- tinal bacterial flora was also reported by Hewitt et al.

(52) after lavage treatment. The difference of endo- toxic activity in lavage- and gut-nonirrigated patients was not the result of a dilution effect by the ortho- grade saline gut-irrigation, since serum sodium, po- tassium, bicarbonate, haematocrit, haemoglobin and body weight were stable before, during and after whole gut-irrigation.

In agreement with our results, Adeyemi et al. (53) found, in patients with active Crohn's disease, elevated elastase/tti-proteinase-inhibitor compared with healthy adults and patients with inactive Crohn's dis- ease. Their results yielded a positive correlation be- tween the elastase/ai-proteinase-inhibitor and the Crohn's disease activity index. In conclusion the lon- gitudinal study showed that

a) elastase/aj-proteinase-inhibitor may be used as an alternative to the Z,/w«/t/s-amoebocyte-lysate-reac- tivity and

b) both tests as well as the Crohn's disease activity index and van Hees activity index yielded a lower degree of endotoxaemia and a reduced inpatient time in lavage patients, compared with gut-non- irrigated patients.

J. Clin. Chem. Clin. Biochem. / Vol. 26,1988 / No. 3

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